Human Akt-3

ABSTRACT

There is disclosed a nucleic acid molecule encoding human Akt-3 protein or a functional equivalent or bioprecursor thereof comprising the amino acid sequence illustrated in Sequence ID No. 3. The human Akt-3 protein itself also forms part of the invention. The nucleic acid molecule and the human Akt-3 protein may themselves be used as medicaments, or in the preparation of medicaments for treating cancer, in their own right or in the form of a pharmaceutically acceptable carrier, diluent or excipient thereof. Further disclosed are methods of identifying agents which influence the activity of a human Akt-3 protein.

This application is a national stage filing of International PublicationNo. WO/00/37613 filed Dec. 17, 1999 which claims priority from GreatBritain Patent Application No. 9828375.7 filed Dec. 22, 1998 andentitled “Human AKT-3”.

FIELD OF THE INVENTION

The present invention is concerned with cloning and expression of a newhuman serine/threonine kinase termed “Akt-3” and, in particular, withnucleic acid molecules encoding the Akt-3 protein, the protein itselfand compounds which can be used to inhibit cell survival.

BACKGROUND OF THE INVENTION

A characteristic feature of many cancer cells is their ability to growindependently of adhesion. In contrast, when untransformed endothelialcells are prevented from adhering to the extracellular matrix (ECM),they undergo apoptosis (Frisch & Francis, 1994; Meredith et al, 1993).The process by which normally adherent cells are triggered to undergoapoptosis when they are unable to adhere to ECM has been termed“anoikis” (Frisch & Ruoslahti, 1997) and is an example of the effect ona cell of removal of a survival factor. Changes in signalling byadhesion molecules can lead to resistance to anoikis (Frisch &Ruoslahti, 1997) and this may contribute to the mechanism whereby cancercells that grow independently of adhesion are able to avoid anoikis.

Akt (also known as protein kinase B (PKB) or “related to A and C proteinkinase” (RAC-PK)) is a serine/threonine kinase that has been implicatedin regulating cell survival (Khwaja et al., 1997; Dudek et al., 1997;Kauffmann-Zeh et al., 1997; Kennedy et al., 1997; Datta et al., 1997;Marte & Downward, 1997). Akt can inhibit apoptosis induced by detachmentfrom ECM (Khwaja et al., 1997), as well as by survival factor withdrawal(Kennedy et al., 1997; Ahmed et al., 1997; Dudek et al., 1997;Kauffman-Zeh et al., 1997; Philpott et al., 1997; Crowder & Freeman,1998; Eves et al., 1998) or irradiation (Kulik et al., 1997).

Akt comprises an NH₂-terminal pleckstrin homology (PH) domain involvedin lipid binding, a kinase domain and a COOH-terminal “tail”. Akt isthought to be activated by recruitment to the plasma membrane andsubsequent phosphorylation by two upstream kinases, PDK-1 and PDK-2(reviewed in Coffer et al, 1998; Alessi & Cohen, 1998). The binding of3-phosphoinositides, generated by phosphatidylinositol 3-kinase (PI3-kinase), to the PH domain of Akt is believed to promote translocationto the plasma membrane and to facilitate phosphorylation of Akt-1 byPDK-1 at Thr³⁰⁸ (Alessi et al., 1996; Alessi et al., 1997; Stephens etal., 1998) or of Akt-2 at Thr³⁰⁹ (Meier et al., 1997). In addition tophosphorylation of Thr³⁰⁸ ₁ full activation requires phosphorylation ofthe COOH tail at Ser⁴⁷³ in Akt-1 (Alessi et al, 1996) or at Ser⁴⁷⁴ inAkt-2 (Meier et al., 1997). The enzyme responsible for phosphorylationof Ser⁴⁷³/Ser⁴⁷⁴ was originally named PDK-2 but recently theintegrin-linked kinase, ILK (Delcommenne et al., 1998) has emerged as acandidate for this function.

Two human isoforms of Akt have been described to date, Akt-1 and Akt-2(Coffer & Woodgett, 1991; Jones et al., 1991; Cheng et al., 1992). Athird isoform, here referred to as Akt-3, has been described in the rat(Konishi et al., 1995). Since this rat Akt-3 possesses an apparentlytruncated tail and thereby lacks Ser⁴⁷³, its regulation may differ fromthat of Akt-1 and Akt-2. Both Akt-1 and Akt-2 are expressed widely,although the expression of Akt-2 is most prominent in insulin-responsivetissues, such as liver and skeletal muscle (Konishi et al., 1994;Altomare et al., 1995). Akt-1 and Akt-2 are activated by insulin in ratadipocytes, hepatocytes and skeletal muscle. In contrast, Akt-3 does notappear to be strongly activated by insulin in these tissues (Walker etal., 1998). The role of the various Akt isoforms in insulin signallingmay limit the utility of compounds that inhibit Akt-1 or Akt-2 activityas such agents may induce symptoms observed in patients with diabetes.We hypothesized that this problem may be avoided by using selectiveinhibitors of Akt-3 and this prompted us to identify the human analogueof rat Akt-3.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have now identified and characterised a nucleicacid molecule that encodes the human isoform of Akt-3. Significantly,human Akt-3 possesses a COOH-terminal tail that contains an amino acidresidue analogous to Ser⁴⁷³/Ser⁴⁷⁴ previously implicated in theactivation of Akt-1/Akt-2, but absent in the rat Akt-3 protein.

Therefore, there is provided by a first aspect of the present inventiona nucleic acid molecule encoding human Akt-3 or a functional equivalent,derivative or bioprecursor thereof, comprising the amino acid sequenceillustrated in FIG. 2 (and as SEQ ID NO:3). Preferably, the molecule isa DNA molecule and even more preferably a cDNA molecule, and even morepreferably comprises the sequence of nucleotides provided in FIG. 1 (SEQID NO:1). Also provided by this aspect of the invention is a nucleicacid molecule capable of hybridising to the molecule according to theinvention under high stringency conditions.

Stringency of hybridisation as used herein refers to conditions underwhich polynucleic acids are stable. The stability of hybrids isreflected in the melting temperature (Tm) of the hybrids. Tm can beapproximated by the formula:81.5° C.+16.6(log₁₀[Na⁺]+0.41 (% G&C)−600/1wherein 1 is the length of the hybrids in nucleotides. Tm decreasesapproximately by 1–1.5° C. with every 1% decrease in sequence homology.

The term “stringency” refers to the hybridisation conditions wherein asingle-stranded nucleic acid joins with a complementary strand when thepurine or pyrimidine bases therein pair with their corresponding base byhydrogen bonding. High stringency conditions favour homologous basepairing whereas low stringency conditions favour non-homologous basepairing.

“Low stringency” conditions comprise, for example, a temperature ofabout 37° C. or less, a formamide concentration of less than about 50%,and a moderate to low salt (SSC) concentration; or, alternatively, atemperature of about 50° C. or less, and a moderate to high salt (SSPE)concentration, for example 1M NaCl.

“High stringency” conditions comprise, for example, a temperature ofabout 42° C. or less, a formamide concentration of less than about 20%,and a low salt (SSC) concentration; or, alternatively, a temperature ofabout 65° C., or less, and a low salt (SSPE) concentration. For example,high stringency conditions comprise hybridization in 0.5 M NaHPO₄, 7%sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C. (Ausubel, F. M. et al.Current Protocols in Molecular Biology, Vol. I, 1989; Green Inc. NewYork, at 2.10.3).

“SSC” comprises a hybridization and wash solution. A stock 20×SSCsolution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.

“SSPE” comprises a hybridization and wash solution. A 1×SSPE solutioncontains 180 mM NaCl, 10 mM NaH₂PO4 and 1 mM EDTA, pH 7.4.

The nucleic acid capable of hybridising to nucleic acid moleculesaccording to the invention will generally be at least 85%, preferably atleast 90% and even more preferably at least 95% homologous to thenucleotide sequences according to the invention.

The DNA molecules according to the invention may, advantageously, beincluded in a suitable expression vector to express polypeptides encodedtherefrom in a suitable host.

The present invention also comprises within its scope proteins orpolypeptides encoded by the nucleic acid molecules according to theinvention or a functional equivalent, derivative or bioprecursorthereof.

An expression vector according to the invention includes a vector havinga nucleic acid according to the invention operably linked to regulatorysequences, such as promoter regions, that are capable of effectingexpression of said DNA fragments. The term “operably linked” refers to ajuxta position wherein the components described are in a relationshippermitting them to function in their intended manner. Such vectors maybe transformed into a suitable host cell to provide for expression of apolypeptide according to the invention. Thus, in a further aspect, theinvention provides a process for preparing polypeptides according to theinvention which comprises cultivating a host cell, transformed ortransfected with an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the polypeptides, and recovering the expressed polypeptides.

The vectors may be, for example, plasmid, virus or phage vectorsprovided with an origin of replication, optionally a promoter for theexpression of said nucleotide and optionally a regulator of thepromoter. The vectors may contain one or more selectable markers, suchas, for example, ampicillin resistance.

Regulatory elements required for expression include promoter sequencesto bind RNA polymerase and transcription initiation sequences forribosome binding. For example, a bacterial expression vector may includea promoter such as the lac promoter and for transcription initiation theShine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryoticexpression vector may include a heterologous or homologous promoter forRNA polymerase II, a downstream polyadenylation signal, the start codonAUG, and a termination codon for detachment of the ribosome. Suchvectors may be obtained commercially or assembled from the sequencesdescribed by methods well known in the art.

A nucleic acid molecule according to the invention may be inserted intothe vectors described in an antisense orientation in order to providefor the production of antisense RNA. Antisense RNA or other antisensenucleic acids may be produced by synthetic means.

In accordance with the present invention, a defined nucleic acidincludes not only the identical nucleic acid but also any minor basevariations including in particular, substitutions in bases which resultin a synonymous codon (a different codon specifying the same amino acidresidue) due to the degenerate code in conservative amino acidsubstitutions. The term “nucleic acid sequence” also includes thecomplementary sequence to any single stranded sequence given regardingbase variations.

The present invention also advantageously provides nucleic acidsequences of at least approximately 10 contiguous nucleotides of anucleic acid according to the invention and preferably from 10 to 120,and even more preferably from 10 to approximately 50 nucleotides. Thesesequences may, advantageously be used as probes or primers to initiatereplication, or the like. Such nucleic acid sequences may be producedaccording to techniques well known in the art, such as by recombinant orsynthetic means. They may also be used in diagnostic kits or the likefor detecting the presence of a nucleic acid according to the invention.These tests generally comprise contacting the probe with the sampleunder hybridising conditions and detecting for the presence of anyduplex or triplex formation between the probe and any nucleic acid inthe sample.

According to the present invention these probes may be anchored to asolid support. Preferably, they are present on an array so that multipleprobes can simultaneously hybridize to a single biological sample. Theprobes can be spotted onto the array or synthesised in situ on thearray. (See Lockhart et al., Nature Biotechnology, vol. 14, December1996 “Expression monitoring by hybridisation to high densityoligonucleotide arrays”). A single array can contain more than 100, 500or even 1,000 different probes in discrete locations.

Advantageously, the nucleic acid sequences, according to the inventionmay be produced using such recombinant or synthetic means, such as forexample using PCR cloning mechanisms which generally involve making apair of primers, which may be from approximately 10 to 50 nucleotides toa region of the gene which is desired to be cloned, bringing the primersinto contact with mRNA, cDNA, or genomic DNA from a human cell,performing a polymerase chain reaction under conditions which bringabout amplification of the desired region, isolating the amplifiedregion or fragment and recovering the amplified DNA. Generally, suchtechniques as defined herein are well known in the art, such asdescribed in Sambrook et al (Molecular Cloning: a Laboratory Manual,1989).

The nucleic acids or oligonucleotides according to the invention maycarry a revealing label. Suitable labels include radioisotopes such as³²P or ³⁵S, enzyme labels or other protein labels such as biotin orfluorescent markers. Such labels may be added to the nucleic acids oroligonucleotides of the invention and may be detected using knowntechniques per se.

A further aspect of the invention comprises human Akt-3 or a functionalequivalent, derivative or bioprecursor thereof, comprising an amino acidsequence as illustrated in FIG. 2.

The polypeptide designated human Akt-3 according to the inventionincludes all possible amino acid variants encoded by the nucleic acidmolecule according to the invention including a polypeptide encoded bysaid molecule and having conservative amino acid changes. Polypeptidesaccording to the invention further include variants of such sequences,including naturally occurring allelic variants which are substantiallyhomologous to said polypeptides. In this context, substantial homologyis regarded as a sequence which has at least 90% amino acid homologywith the polypeptides encoded by the nucleic acid molecules according tothe invention and even more preferably at least 95% amino acid homology.

The nucleic acid molecule or the human Akt-3 according to the inventionmay, advantageously, be used as a medicament or in the preparation of amedicament, for treating disease associated with Akt-3 activity such as,cancer or the like.

Advantageously, the nucleic acid molecule or the polypeptide accordingto the invention may be provided in a pharmaceutical compositiontogether with a pharmaceutically acceptable carrier, diluent orexcipient therefor.

The present invention is further directed to inhibiting Akt-3 in vivo bythe use of antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the mature proteinsequence, which encodes for the protein of the present invention, isused to design an antisense RNA oligonucleotide of from 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple-helix—see Leeet al. Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456(1988); and Dervan et al., Science, 251: 1360 (1991), thereby preventingtranscription and the production of Akt-3. The antisense RNAoligonucleotide hybridises to the mRNA in vivo and blocks translation ofan mRNA molecule into the Akt-3 (antisense—Okano, J. Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1998)).

Alternatively, the oligonucleotide described above can be delivered tocells by procedures in the art such that the anti-sense RNA or DNA maybe expressed in vivo to inhibit production of Akt-3 in the mannerdescribed above.

Antisense constructs to Akt-3, therefore, may inhibit the survival ofthe cell and prevent further cancer or tumour growth.

According to a further aspect of the invention there is also provided atransgenic cell, tissue or organism comprising a transgene capable ofexpressing human Akt-3 protein according to the invention. The term“transgene capable of expression” as used herein means a suitablenucleic acid sequence which leads to expression of human Akt-3 or humanproteins having the same function and/or activity. The transgene, mayinclude, for example, genomic nucleic acid isolated from human cells orsynthetic nucleic acid, including DNA integrated into the genome or inan extrachromosomal state. Preferably, the transgene comprises thenucleic acid sequence encoding the proteins according to the inventionas described herein, or a functional fragment of said nucleic acid. Afunctional fragment of said nucleic acid should be taken to mean afragment of the gene comprising said nucleic acid coding for theproteins according to the invention or a functional equivalent,derivative or a non-functional derivative such as a dominant negativemutant, or bioprecursor of said proteins. For example, it would bereadily apparent to persons skilled in the art that nucleotidesubstitutions or deletions may be used using routine techniques, whichdo not affect the protein sequence encoded by said nucleic acid, orwhich encode a functional protein according to the invention.

Human Akt-3 protein expressed by said transgenic cell, tissue ororganism or a functional equivalent or bioprecursor of said protein alsoform part of the present invention.

Antibodies to human Akt-3 may, advantageously, be prepared by techniqueswhich are known in the art. For example, polyclonal antibodies may beprepared by inoculating a host animal, such as a mouse, with human Akt-3according to the invention or an epitope thereof and recovering immuneserum. Monoclonal antibodies may be prepared according to knowntechniques such as described by Kohler R. and Milstein C., Nature (1975)256, 495–497.

Antibodies according to the invention may also be used in a method ofdetecting for the presence of human Akt-3 according to the invention,which method comprises reacting the antibody with a sample andidentifying any protein bound to said antibody. A kit may also beprovided for performing said method which comprises an antibodyaccording to the invention and means for reacting the antibody with saidsample.

Proteins which interact with the polypeptide of the invention may beidentified by, for example, investigating protein—protein interactionsusing the two-hybrid vector system first proposed by Chien et al (1991).Proc. Natl. Acad. Sci. USA 88: 9578–9582.

This technique is based on functional reconstitution in vivo of atranscription factor which activates a reporter gene. More particularlythe technique comprises providing an appropriate host cell with a DNAconstruct comprising a reporter gene under the control of a promoterregulated by a transcription factor having a DNA binding domain and anactivating domain, expressing in the host cell a first hybrid DNAsequence encoding a first fusion of a fragment or all of a nucleic acidsequence according to the invention and either said DNA binding domainor said activating domain of the transcription factor, expressing in thehost at least one second hybrid DNA sequence, such as a library or thelike, encoding putative binding proteins to be investigated togetherwith the DNA binding or activating domain of the transcription factorwhich is not incorporated in the first fusion; detecting any binding ofthe proteins to be investigated with a protein according to theinvention by detecting for the presence of any reporter gene product inthe host cell; optionally isolating second hybrid DNA sequences encodingthe binding protein.

An example of such a technique utilises the GAL4 protein in yeast. GAL4is a transcriptional activator of galactose metabolism in yeast and hasa separate domain for binding to activators upstream of the galactosemetabolising genes as well as a protein binding domain. Nucleotidevectors may be constructed, one of which comprises the nucleotideresidues encoding the DNA binding domain of GAL4. These binding domainresidues may be fused to a known protein encoding sequence, such as forexample the nucleic acids according to the invention. The other vectorcomprises the residues encoding the protein binding domain of GAL4.These residues are fused to residues encoding a test protein. Anyinteraction between polypeptides encoded by the nucleic acid accordingto the invention and the protein to be tested leads to transcriptionalactivation of a reporter molecule in a GAL-4 transcription deficientyeast cell into which the vectors have been transformed. Preferably, areporter molecule such as β-galactosidase is activated upon restorationof transcription of the yeast galactose metabolism genes.

A further aspect of the invention provides a method of identifyingcompounds which selectively inhibit human Akt-3 mediated promotion ofcell survival said method comprising i) providing a cell transformedwith an expression vector activating the Akt-3 pathway which cellsurvives in the presence or absence of a survival factor compared to acontrol cell which has not been transformed with said vector and willdie in the absence of said survival factor ii) contacting said cellswith a test compound following removal of said cells from said survivalfactors, wherein death of said transformed cell is indicative ofselective inhibition of said compound on the survival promoting humanAkt-3 pathway.

Alternatively, the survival promoting activity of Akt-3 could beassessed by i) providing a cell transformed with an expression vectoractivating the Akt-3 pathway in addition to a control cell which has notbeen transformed with said vector, ii) contacting each of said cellswith a death inducing agent, whereby death of said control cell andsurvival of said transformed cell is indicative of the survivalpromoting activity of the activated Akt-3 pathway, iii) subsequentlycontacting said transformed cell without removal of said death inducingagent, with a test compound, wherein death of said cell is indicative ofselective inhibition of said compound on the survival promoting humanAkt-3 pathway.

In a further aspect the present invention provides methods to identifyagents that affect the activity of the human Akt-3 protein, comprisingcontacting said protein with a substrate, regulatory molecule orsurrogate thereof and monitoring the interaction with the test substanceusing standard phosphorylation or binding assays well known in the art.

Compounds which are identified according to this aspect of the inventionin addition to antibodies to the human Akt-3 may, advantageously, beutilised as a medicament or alternatively in the preparation of amedicament for treating diseases associated with expression of humanAkt-3 protein according to the invention.

A further aspect of the invention provides a pharmaceutical compositioncomprising any of a compound, an antisense molecule or an antibodyaccording to the invention together with a pharmaceutically acceptablecarrier, diluent or excipient therefor.

The antisense molecules or indeed the compounds identified as agonistsor antagonists of the nucleic acids or polypeptides according to theinvention may be used in the form of a pharmaceutical composition, whichmay be prepared according to procedures well known in the art. Preferredcompositions include a pharmaceutically acceptable vehicle or diluent orexcipient, such as for example, a physiological saline solution. Otherpharmaceutically acceptable carriers including other non-toxic salts,sterile water or the like may also be used. A suitable buffer may alsobe present allowing the compositions to be lyophilized and stored insterile conditions prior to reconstitution by the addition of sterilewater for subsequent administration. Incorporation of the polypeptidesof the invention into a solid or semi-solid biologically compatiblematrix may be carried out which can be implanted into tissues requiringtreatment.

The carrier can also contain other pharmaceutically acceptableexcipients for modifying other conditions such as pH, osmolarity,viscosity, sterility, lipophilicity, solubility or the like.

Pharmaceutically acceptable excipients which permit sustained or delayedrelease following administration may also be included.

The polypeptides, the nucleic acid molecules or compounds according tothe invention may be administered orally. In this embodiment they may beencapsulated and combined with suitable carriers in solid dosage formswhich would be well known to those skilled in the art.

As would be well known to those of skill in the art, the specific dosageregime may be calculated according to the body surface area of thepatient or the volume of body space to be occupied, dependent upon theparticular route of administration to be used. The amount of thecomposition actually administered will, however, be determined by amedical practitioner, based on the circumstances pertaining to thedisorder to be treated, such as the severity of the symptoms, thecomposition to be administered, the age, weight, and response of theindividual patient and the chosen route of administration.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more clearly understood with reference tothe following example which is purely exemplary and the accompanyingdrawings wherein:

FIG. 1 is an illustration of the cDNA sequence (SEQ ID NO: 1) anddeduced amino acid sequence (SEQ ID NO: 3) of human Akt-3. The Akt-3coding sequence and parts of the 5′ and 3′ untranslated regions areshown and numbered in the left hand column. The deduced amino acidsequence of the Akt-3 protein is shown above the corresponding DNAsequence and is numbered in the right hand column. The two amino acidresidues that are presumed to be phosphorylated upon activation of Akt-3(Thr³⁰⁵ and Ser⁴⁷² are in bold and marked with an asterisk. TheCOOH-terminal part of the human Akt-3 protein that differs with the rathomologue is underlined.

FIG. 2 is an alignment of the deduced amino acid sequences for humanAkt-1 (SEQ ID NO: 15), Akt-2 (SEQ ID NO: 16) and Akt-3 (SEQ ID NO: 3).The sequences were aligned using the ClustalW alignment program (EMBL,Heidelberg, Germany). Amino acid residues conserved between all threeproteins are included in the black areas. Residues conserved betweenonly two of the sequences are shaded in grey. Amino acid residues arenumbered in the right hand column. The conserved Thr and Ser residuesthat are presumed to be phosphorylated upon activation are marked withan asterisk above the sequence.

FIG. 3 is an illustration of phosphorylation of histone H2B by Akt-3variants. (A) Akt-3 was expressed as a GST fusion protein in E. Coli. Toassess hAkt-3 activity, Histone H2B was incubated with GST-Akt-3 andGST-Akt-3 variants for the indicated time and the extent ofphosphorylation assessed after SDS-PAGE. The variants of Akt-3 aredesignated: W.T., wild type; T305D, Thr³⁰⁵ mutated to Asp; S472D, Ser⁴⁷²mutated to Asp; T305D,S472D, both Thr³⁰⁵ and Ser⁴⁷² mutated to Asp. Nosignificant phosphorylation was observed when GST was used in place ofGST-Akt. The results are the mean (±s.e.m.; n=3 to 6) and are expressedrelative to the extent of phosphorylation of H2B catalysed by T305D,S472D hAkt-3 after 45 minutes. (B) HEK-293 cells were transfected witheither vector (lanes 1 & 2) or Akt-3 (lanes 3 and 4) AKt-3T305A (lanes 5& 6) or Akt-3 S472A (lanes 7 and 8) and either treated with buffer(lanes 1, 3 5 and 7) or IGF-1 (50 ng/ml; lanes 2, 4, 6 and 8). Akt-3 wasimmunoprecipitated with antibody 3F10 (anti-HA tag). Samples wereanalysed by blotting for the HA-tag (upper panel) or with aphosphospecific antibody which recognises phosphorylated ser⁴⁷² (lowerpanel). (C) Akt activity in HA-immuno-precipitates from samples preparedas described above was assessed by measuring phosphorylation of apeptide substrate (Crosstide). The results are expressed as the increasein activity compared to unstimulated cells transfected with empty vector(mean±s.e.m., n=7).

FIG. 4 is an illustration of inhibition of Akt-3 by staurosporine and RO31-8220. Histone H2B was treated with Akt-3 (T305D,S472D variant) in thepresence of the indicated concentrations of either staurosporine or RO31-8220. After 30 minutes, the reaction was terminated and the extent ofH2B phosphorylation quantified on a phosphorimager following SDS-PAGE.The results (mean±s.e.m., n=3) are expressed as relative to (%) thephosphorylation observed in the presence of solvent (control, “C”).

FIG. 5 is an illustration of chromosomal localisation of human Akt-3.(A) Diagram of FISH mapping results of Akt-3. Each dot represents thedouble FISH signals detected on human chromosome 1, region q43–q44. (B)Example of FISH mapping of Akt-3. The left panel shows the FISH signalson chromosome 1. The right panel shows the same mitotic figure stainedwith 4′,6-diamdino-2-phenylindole to identify chromosome 1.

FIG. 6 is an illustration of expression of Akt-3 in different humantissues. (A) Northern blot analysis of tissue expression of Akt-3. Theexpression of hAkt-3 mRNA in different human tissues was assessed usinga probe corresponding to the 3′ untranslated region of hAkt-3 to analysea blot of human polyA⁺ RNA (AMultiple Tissue Northern≅). Human β-actinwas used as a control to confirm equal loading of the lanes (data notshown). (B) and (C) RT-PCR analysis of tissue expression of Akt-3.RT-PCR analyses were performed on cDNA from different human tissues (B)and from different tumor cell lines (C) using primers specific for humanAkt-3 or G3PDH (control) for the indicated number of PCR cycles. Bandsof the expected size (425 bp for Akt-3 and 1 kb for G3PDH) are visibleon the gels. The images from the ethidium bromide stained 1.2% agarosegels were inverted for clarity using the EagleSight software(Stratagene). The results from similar PCR reactions performed for 25,30 or 35 cycles are not shown but indicated that the results from thisfigure are in the linear range of amplification. Caco-2=colorectaladenocarcinoma; T-84=colorectal carcinoma; MCF-7=breast adenocarcinoma;T-47D=breast ductal gland carcinoma; HT1080=bone fibrosarcoma;SaOS-2=osteosarcoma; SK-N-MC=neuroblastoma; HepG2=hepatoblastoma;JURKAT=T-cell leukemia.

FIG. 7 is an illustration of the results obtained by scintillationcounting in a scintillation proximity assay to identify agents thatmodulate the activity of Akt-3 activity.

FIG. 8 is an illustration of the results obtained from an Akt-3 filterassay to identify agents that modulate activity of Akt-3.

EXAMPLES

Materials and Methods

Oligonucleotide Synthesis and DNA Sequence Determination

All primers were obtained from Eurogentec, Seraing, Belgium.Insert-specific sequencing primers (15- and 16-mers) were designed byvisual inspection of the DNA sequences. DNA was prepared onQiagen-tip-20 columns or on Qiaquick spin columns (Qiagen GmbH,Düsseldorf, Germany) and recovered from the spin columns in 30 FlTris/EDTA-buffer (10 mM Tris HCl pH 7.5, 1 mM EDTA (sodium salt)).Sequencing reactions were performed using BigDye™ Terminator CycleSequencing Ready Reaction kits (Perkin Elmer, ABI Division, Foster City,Calif., USA) and were run on an Applied Biosystems 377 DNA sequencer(Perkin Elmer, ABI Division, Foster City, Calif., USA).

Molecular Cloning of Human Akt-3.

Using the rat RAC-PKγ sequence (Konishi et al, 1995; GenBank acc. No.D49836) as a query sequence, a BLAST (Basic Local Alignment Search Tool;Altschul et al., 1990) search was carried out in the WashU Merckexpressed sequence tag (EST) database (Lennon et al., 1996) and in theproprietary LifeSeq™ human EST database (Incyte Pharmaceuticals Inc,Palo Alto, Calif., USA). Several human EST clones with high similarityto the rat RAC-PKγ were identified. One EST sequence (Incyte accessionnumber 2573448) derived from a hippocampal cDNA library, contained partof the coding sequence including the putative methionine start codon(ATG) and part of the 5′ untranslated region. The start codon wassurrounded by a Kozak consensus sequence for translation start and anin-frame stop codon was present at positions −6 to −3. Based on this 239bp sequence, oligonucleotide sense primers were synthesised for 3′ rapidamplification of cDNA ends (3′ RACE) experiments: Akt-3sp1=5′-ACC ATTTCT CCA AGT TGG GGG CTC AG-3′ (SEQ ID NO: 4) and Akt-3sp2=5′GGG AGT CATCAT GAG CGA TGT TAC C-3′ (SEQ ID NO: 5). 3′RACE experiments wereperformed on human fetal brain or human cerebellum Marathon-Ready™ cDNA(Clontech Laboratories, Palo Alto, Calif., USA) according tomanufacturer's instructions using Akt-3sp1/race-ap1 as primers in theprimary PCR and Akt-3sp2/race-ap2 in the nested PCR. Resulting PCRfragments were cloned and sequenced. This extended the Akt-3 codingsequence by 916 bp, but the novel sequence did not include an in-framestop codon. A second round of 3′ RACE amplification was performed onhuman brain Marathon Ready™ cDNA using sense primers based on thesequence obtained in the first round (Akt-3sp3=5′CAC TCC AGA ATA TCT GGCACC AGA GG-3′ (SEQ ID NO: 6) and Akt-3sp4=5′CTA TGG CCG AGC AGT AGA CTGGTG G-3′) (SEQ ID NO: 7) in combination with race-ap1 and race-ap2,respectively. The sequence obtained included an in-frame stop codon andthe 3′ untranslated sequence up to the poly(A) tail. Antisense primerswere designed based on the 3′ untranslated region (Akt-3ap4=5′-TGC CCCTGC TAT GTG TAA GAG CTA GG-3′(SEQ ID NO: 8) and Akt-3ap5=5′ AAG AGC TAGGAC TGG TGA TGT CCA GG-3′) (SEQ ID NO: 9) and the complete Akt-3 codingsequence was amplified from human hippocampal cDNA usingAkt-3sp1/Akt-3ap4 (primary PCR) and Akt-3sp2/Akt-3ap5 (nested PCR) asprimers. The resulting 1200 bp PCR fragment was then cloned in theTA-cloning vector pCR2.1 (original TA cloning kit, Invitrogen BV, Leek,The Netherlands) and the inserts of several clones were completelysequenced. One clone containing an insert with the confirmed sequence(hAkt-3/pCR2.1) was used for subsequent subcloning to the mammalianexpression vector pcDNA-3 (Invitrogen), yielding constructhAkt-3/pcDNA-3. In order to make a construct coding for a COOH-terminaltagged Akt-3 protein, a fragment of 553 bp was amplified from plasmidAkt-3/pcDNA-3 using an antisense primer incorporating a XhoI restrictionsite and the sequence coding for a hemagglutinin (HA) tag (YPYDVPDYA)(SEQ ID NO: 13) after amino acid 479 of the Akt-3 sequence. Thisfragment was recloned into plasmid hAkt-3/pcDNA-3 using BstEII and XhoIrestriction sites yielding construct HA-hAkt-3/pcDNA-3.

Constructs and Mutants for E. coli Expression of Akt-3.

In order to express the human Akt-3 protein in E. coli, the completeAkt-3 coding sequence was amplified from plasmid hAkt-3/pCR2.1 usingprimers introducing a EcoRI restriction site and a XhoI restriction siteat the 5′ and 3′ ends, respectively. This PCR fragment was cloned as aEcoRI/XhoI fragment in vector pGEX-4T-3 (Amersham Pharmacia Biotech,Uppsala, Sweden) yielding construct hAKT-3(WT)/pGEX-4T-3, and thesequence of the insert was confirmed by sequence analysis.

Mutants of this construct were made using the Quickchange site-directedmutagenesis kit (Stratagene, La Jolla, Calif., USA) according to themanufacturer=s instructions. The T305D mutant (constructhAKT-3(T305D)/pGEX-4T-3) was created by mutating ACA at position 923–925to GAC, resulting in a Thr³⁰⁵ to Asp mutation in the resulting protein.The S472D mutant (construct hAKT-3(S472D)/pGEX-4T-3) was created bychanging TC at position 1404–1405 to GA using PCR with a long antisenseprimer incorporating the change, resulting in a Ser⁴⁷² to Asp mutationin the resulting protein. A double mutant was also constructed bysite-directed mutagenesis on hAKT-3(S472D)/pGEX-4T-3 and contained boththese mutations (construct hAKT-3(T305D/S472D)/pGEX-4T-3). The insertsof all resulting constructs were confirmed by complete sequenceanalysis. The fusion proteins resulting from expression of theseconstructs in E. coli contain a GST moiety coupled to the NH₂-terminusof the human Akt-3 sequence.

Expression in Cos-7 Cells and HEK-293 Cells

Akt-3 was transiently expressed in Cos-7 by calcium phosphatetransfection of the cells with the construct HA-hAkt-3/pcDNA-3. Thecells were stimulated with 10 ng/ml IGF-1 for 30 minutes, lysed andAkt-3 immunoprecipitated with mAb 12CA5. Akt-3 activity was assessed asdescribed below.

For expression in HEK-293 cells, cells were transfected with pCDNA-3Akt-3 constructs as described previously (Alessi et al 1996). Afterstimulation with IGF, the cells were lysed (Alessi et al 1996) andHA-Akt immunoprecipitated with antibody 3F10 (Roche MolecularBiochemicals). Akt activity was assessed in immune complexes bymeasuring phosphorylation of a peptide substrate (Crosstide) in thepresence of 1 μM PKI (PKA inhibitor) and 1 μM GF 109302× (PKC inhibitor)as described.

Expression and Assay of Wild-Type and Mutant Akt-3 in E. coli.

The pGEX expression constructs were transformed into E. coli strain BL21DE3 and GST-fusion proteins of wild-type and mutated Akt-3 were purifiedon glutathione sepharose according to the manufacturers instructions(Amersham Pharmacia Biotech, Uppsala, Sweden). The protein eluted fromthe beads was stored in 50% glycerol at −20° C. Akt activity wasassessed by incubating 0.8 Fg of the purified enzyme for 30 minutes atroom temperature (unless otherwise indicated) in a buffer containing 10mM HEPES, 10 mM MgCl₂, 1 mM DTT, 0.1 mg/ml histone H2B at pH 7.0, in atotal volume of 25 Fl and containing 10 FCi [γ-³²P]-ATP (6000 Ci/mmol).Initial experiments indicated that the reaction was linear with time forat least 45 minutes. The reaction was stopped by the addition of 25 Flsample buffer for SDS-PAGE. The results were quantified on aphosphorimager following SDS-PAGE on a 15% (w/v) acrylamide gel.

Chromosomal Mapping Studies

Chromosomal mapping studies were carried out by SeeDNA Biotech Inc,Toronto, Canada using fluorescent in situ hybridisation (FISH) analysisessentially as described (Heng et al., 1992; Heng & Tsui, 1993).Briefly, human lymphocytes were cultured at 37° C. for 68–72 h beforetreatment with 0.18 mg/ml 5-bromo-2′-deoxyuridine (BrdU) to synchronizethe cell cycle in the cell population. The synchronized cells werewashed and recultured at 37° C. for 6 h. Cells were harvested and slideswere prepared using standard procedures including hypotonic treatment,fixation and air-drying. A cDNA probe for Akt-3 (1.44 kb EcoRI fragmentof clone hAkt-3/pcDNA-3) was biotinylated and used for FISH detection.Slides were baked at 55° C. for 1 h, treated with Rnase and denatured in70% (v/v) formamide in 2× NaCl/Cit (0.3 M NaCl, 0.03 M disodium citrate,pH 7.0) for 2 min at 70EC followed by dehydration in ethanol. Probeswere denatured prior to loading on the denatured chromosomal slides.After overnight hybridisation, slides were washed and FISH signals andthe 4′,6-diamidiono-2-phenylindole banding pattern were recordedseparately on photographic film, and the assignment of the FISH mappingdata with chromosomal bands was achieved by superimposition of FISHsignals with 4,6-diamidino-2-phenylindole banded chromosomes (Heng &Tsui, 1993).

Northern Blot Analysis.

Northern blots containing 2 Fg of poly(A)-rich RNA derived fromdifferent human tissues (Clontech Laboratories, Palo Alto, Calif., USA)were hybridised according to the manufacturer's instructions with aα-³²P-dCTP random-priming labelled (HighPrime kit, Boehringer Mannheim)454 bp NotI-XbaI Akt-3 fragment (nucleotides 1404 to 1857) correspondingto part of the 3′ untranslated sequence.

Reverse Transcription (RT)-PCR Analysis

Oligonucleotide primers were designed for the specific PCR amplificationof a fragment from Akt-3. These primers were Akt-3sp2=5′-GGG AGT CAT CATGAG CGA TGT TAC C-3′ (SEQ ID NO: 5) (sense primer) and Akt-3ap1=5′-GGGTTG TAG AGG CAT CCA TCT CTT CC-3′ (SEQ ID NO: 11) (antisense primer),yielding a 425 bp product. PCR amplifications for humanglyceraldehyde-3-phosphate dehydrogenase (G3PDH) were performed on thesame cDNA samples as positive controls using G3PDH primers 5′-TGA AGGTCG GAG TCA ACG GAT TTG GT-3′(SEQ ID NO: 10) (sense primer) and 5′-CATGTG GGC CAT GAG GTC CAC CAC-3′ (SEQ ID NO: 14) (antisense primer),yielding a 1000 bp fragment. These primers were used for PCRamplifications on Multiple Tissue cDNA panels (Clontech Laboratories)and on cDNA prepared from tumor cell lines. For the preparation of tumorcell cDNA, cells were homogenised and total RNA prepared using theRNeasy Mini kit (Qiagen GmbH, Hilden, Germany) according tomanufacturer's instructions. 1 Fg of total RNA was reverse transcribedusing oligo(dT)₁₅ as a primer and 50 U of Expand™ Reverse Transcriptase(Boehringer Mannheim, Mannheim, Germany) according to the manufacturer'sinstructions. PCR reactions with Akt-3-specific or G3PDH-specificprimers were then performed on 1 Fl of cDNA. Images of the ethidiumbromide stained gels were obtained using the Eagle Eye II Video system(Stratagene, La Jolla, Calif., USA) and PCR bands analysed using theEagleSight software.

Assays to Identify Agents that Modulate the Activity of Akt-3

To identify agents that modulate the activity of Akt-3, SPA(scintillation proximity assay) and filter assays for Akt-3 activitywere developed.

SPA assays were performed at 25° C. for 3 hrs in the presence of 25 mMHepes, pH 7.0, containing 15 mM MgCl₂, 1 mM DTT. Each assay wasperformed in a 100 Fl reaction volume containing 111 nM GST-AKT-3(diluted in 25 mM Hepes, pH 7.0, containing 15 mM MgCl₂, 1 mM DTT), 0.75FM Biotinylated Histone H2B, 2 nM [γ-³³P]-ATP and any agents under test.The reaction was terminated by addition of 100 Fl Stop mix (50 FM ATP, 5mM EDTA, 0.1% BSA, 0.1% Triton X-100 and 7.5 mg/ml Streptavidin coatedPVT SPA beads). After allowing the beads to settle for 30 minutes, theassay mixture was counted in a microtiterplate scintillation counter.The results are illustrated in FIG. 7.

AKT3 filter assays were performed at 25° C. for 3 hrs in the presence of25 mM Hepes, pH7.0, containing 15 mM MgCl₂, 1 mM DTT. Each assay wasperformed in a 100 Fl reaction volume containing 111 nM GST-AKT-3(diluted in 25 mM Hepes, pH7.0, containing 15 mM MgCl₂, 1 mM DTT), 2.5FM Histone H2B, 2 nM [γ-³³P]-ATP and any agents under test. The reactionwas terminated by addition of 100 Fl 75 mM H₃PO₄. 90 Fl of the assaymixture was filtered through Phosphocellulose cation exchange paper.After five times washing with 75 FM H₃PO₄, the filterpaper was countedin a microtiterplate scintillation counter. The results are illustratedin FIG. 8.

Results

Molecular Cloning of Human Akt-3.

Similarity searching of the LifeSeq™ and EMBL databases using the ratAkt-3 sequence as a query sequence yielded several human EST sequenceswhich encoded part of the human homologue of rat Akt-3. Using the DNAsequence information in the databases, we were able in subsequent 3′RACE experiments to deduce the complete cDNA sequence for the humanAkt-3 (FIG. 1) (SEQ ID NO:1, coding DNA is provided as SEQ ID NO:2). Theobtained cDNA sequence encoded a protein of 479 amino acid residues (SEQID NO:3) with a calculated molecular mass of 55770 Da. The first 451amino acids of the human Akt-3 protein contain only two differences tothe corresponding rat sequence (Konishi et al., 1995)—Asp (rat) to Gly(human) at position 10 and Pro (rat) to Ala (human) at position 396 andencode a pleckstrin homology domain, a kinase domain and a COOH-terminal“tail”. The two amino acid residues that are presumed to bephosphorylated upon activation of Akt-3 (Thr³⁰⁵ and Ser⁴⁷²) are in boldand marked with an asterisk. The COOH-terminal part of the human Akt-3protein that differs with the rat homologue extends from amino acid 452through amino acid 479. The predicted Akt-3 (FIG. 2) protein showssignificant similarity with Akt-1 (Jones et al, 1991; 83.6% identity;87.8% similarity) and with Akt-2 (Cheng et al., 1992; 78% identity;84.3% similarity). The COOH-terminal ‘tail’ has been observed in bothhuman and rat Akt-1 and Akt-2 proteins, but it is apparently truncatedin the only other reported Akt-3 sequence (rat Akt-3, Konishi et al.,1995; accession number D49836). 3′RACE experiments performed on humancDNAs derived from different tissues did not yield evidence for theexistence of a shorter form of Akt-3 that would be analogous to the ratAkt-3 (data not shown). The tail in human Akt-3 comprises 28 amino acidresidues (YDEDGMDCMDNERRPHFPQFSYSASGRE) (SEQ ID NO: 12) that replace 3amino acid residues in the rat sequence (CPL). The tail in human Akt-3contains a serine residue at position 472 (shown in bold) thatcorresponds to Ser⁴⁷³ in Akt-1 or Ser⁴⁷⁴ in Akt-2. Phosphorylation ofSer⁴⁷³ and Ser⁴⁷⁴ has previously been implicated in the activation ofAkt-1 and Akt-2, respectively (Alessi et al., 1996; Meier et al., 1997).Thr³⁰⁸ (in the kinase domain) has also been implicated in the activationof Akt-1 and this residue is also conserved in human Akt-3 (Thr³⁰⁵).

Characterisation of Akt-3 Activity.

To characterise the enzymatic activity of Akt-3, we expressed andpurified the recombinant enzyme as a GST fusion protein. Analysis of thepurified product by SDS-PAGE indicated the protein was apparently >90%pure. The purified enzyme was able to phosphorylate histone H2B (FIG.3), and no phosphorylation was observed using recombinant GST alone.Previously, the enzymatic activity of Akt-1 has been shown to beincreased by phosphorylation of Thr³⁰⁸ and Ser⁴⁷³, and mutation of boththese residues to Asp (to mimic phosphorylation) synergisticallyactivates Akt-1 (Alessi et al., 1996). To investigate whether Akt-3 issimilarly regulated, GST-fusion proteins in which either Thr³⁰⁵ orSer⁴⁷² (corresponding to Thr³⁰⁸ and Ser⁴⁷³ in Akt-1) or both Thr³⁰⁵ andSer⁴⁷² had been mutated to Asp were expressed and assayed in comparisonto the wild-type enzyme. Mutation of Thr³⁰⁵ to Asp (“T305D”) resulted ina 2.0-fold increase in the initial rate of phosphorylation of histoneH2B, whereas mutation of Ser⁴⁷² to Asp(S472D”) increased the initialrate only 1.4 fold (FIG. 3A). When both Thr³⁰⁵ and Ser⁴⁷² (“T305D,S472D)were mutated to Asp, a 3.2-fold increase in the initial phosphorylationrate was observed.

To confirm that extracellular stimuli can activate Akt-3 in mammaliancells, Cos-7 cells were transfected with a cDNA encoding Akt-3 fused toa HA tag. Akt-3 activity in HA immunoprecipitates was increased 1.5 and1.9 fold (n=2) following stimulation with IGF-1 (10 ng/ml).

To further confirm that extracellular stimuli can activate Akt-3 inmammalian cells, HEK-293 cells were transfected with a cDNA encodingAkt-3 fused to a HA epitope tag. Upon treatment with IGF, Akt-3 activityin anti-HA immunoprecipitates (FIG. 3B) was increased almost 60-foldabove that in untransfected cells (FIG. 3C). Akt variants in whichThr³⁰⁵ and Ser⁴⁷² were mutated to alanine were refractory to activationby IGF. Consistent with this, Western blotting with a Ser⁴⁷²phosphospecific antibody of HA immunoprecipitates from cells stimulatedwith IGF demonstrated that Ser⁴⁷² was phosphorylated followingstimulation with IGF (FIG. 3B). In addition, activation of Akt-3 wasinhibited by prior treatment with CY29 4002 (100 FM, 94% inhibition),data not shown).

To characterise human Akt-3 further, we investigated the ability of arange of Ser/Thr kinase inhibitors to inhibit Akt-3. These included Go6976, GF-109203× (both protein kinase C (PKC) inhibitors); H-85, H-88,H-89 and KT5720 (protein kinase A (PKA) inhibitors), KN-62(Ca⁺²/Calmodulin dependent kinase inhibitor) and PD 98059 (MEKinhibitor). When tested at a concentration of 10 FM these compounds hadno significant effect on the activity of the T305D,S472D variant ofAkt-3. However, the broad spectrum kinase inhibitor staurosporine(IC₅₀=2.0±0.3 FM) and the PKC inhibitor Ro 31-8220 (IC₅₀=3.2±1.0 FM)inhibited the T305D,S472D variant of Akt-3 (FIG. 4).

Chromosomal Localisation of Akt-3.

The complete coding sequence of Akt-3 was used as a probe for FISHanalysis. Under the conditions used, the hybridisation efficiency wasapproximately 75% for this probe (among 100 checked mitotic figures, 75of them showed signals on one pair of the chromosomes). Since theDAPI-banding was used to identify the specific chromosome, theassignment between the signal from the probe and the long arm ofchromosome 1 was obtained. The detailed position was further determinedin the diagram based upon the summary from 10 photographs (FIG. 5A).There was no additional locus picked by FISH detection under theconditions used, therefore, it was concluded that Akt-3 is located athuman chromosome 1, region q43–q44. An example of the mapping results ispresented in FIG. 5B.

Tissue Distribution of Akt-3 mRNA.

Northern blot analysis was performed on mRNA derived from differenthuman tissues. Akt-3 mRNA was detected as two transcripts ofapproximately 4.5 kb and 7.5 kb, showing similar patterns of expression(FIG. 6A). Akt-3 mRNA was expressed in a range of tissues, mostprominently in brain. Similarly, rat Akt-3 was detected as multipletranscripts most highly expressed in brain (Konishi et al., 1995). Theweakest expression of Akt-3 was observed in two insulin-responsivetissues, skeletal muscle and liver. Akt-3 was also expressed in a numberof cancer cell lines including SW480 colorectal adenocarcinoma, A549lung carcinoma and G361 Melanoma (data not shown).

To confirm the Northern blot analysis, PCR reactions were performed withAkt-3 specific and G3PDH-specific (internal control) primers on cDNAsderived from different human tissues (FIG. 6B). The Akt-3 message waspresent in every tissue tested, since a specific 425 bp fragment wasamplified in every cDNA after 30 cycles of PCR. Akt-3 mRNA expressionwas high in placenta, ovary and spleen. Moderate expression was seen inbrain, heart, kidney, colon, prostate, small intestine and testis.Lowest expression was in liver, lung, pancreas, skeletal muscle,peripheral blood leukocytes and thymus. In tumor cell lines (FIG. 6C),Akt-3 mRNA expression was relatively high in HT-1080 bone fibrosarcomacells, in SaOS-2 osteosarcoma and in JURKAT T-cell leukemia cells (Akt-3band detectable after 30 cycles of PCR). Caco-2 colorectaladenocarcinoma, T84 colorectal carcinoma, MCF-7 breast adenocarcinomaand SK-N-MC neuroblastoma cells show Akt-3 mRNA expression after 35cycles of PCR. In T-47D breast ductal gland carcinoma and HepG2hepatoblastoma, expression of Akt-3 mRNA is very low or absent (nosignal detectable after 35 cycles of PCR).

Akt-1 and Akt-2 have been identified in several species. Human (Jones etal., 1991; Coffer et al 1991), mouse (Bellacosa et al., 1993) and bovine(Coffer & Woodgett, 1991) Akt-1 clones have been reported, whereas human(Cheng et al., 1992) mouse (Altomare et al., 1995) and rat (Konishi etal., 1994) clones of Akt-2 have been identified. However, Akt-3 has onlybeen previously identified in rat (Konishi et al, 1995). The presentinventors have identified the human isoform of Akt-3. Although humanAkt-3 shows considerable similarity to human Akt-1 and Akt-2, thediscovery of human Akt-3 is particularly significant because the cDNAsequence encodes a COOH-terminal Atail≅ which includes a phosphorylationsite implicated in the activation of Akt-1 and Akt-2 (Alessi et al.,1996; Meier et al., 1997). This tail is absent from the predicted ratamino acid sequence. Human Akt-3 appears to be activated byphosphorylation in a similar fashion as Akt-1 and Akt-2. However, itsexpression profile suggests that the principal function of this enzymeis not in regulating responses to insulin.

The sequence which has been identified represents the human homologue ofAkt-3. This assignment is based on the >99% identity between the rat andhuman Akt-3 protein sequences. With the exception of the COOH-terminaltail seen in human Akt-3, there are only 2 amino acid differences (Gly¹⁰and Ala³⁹⁶ in human Akt-3) between the rat and human Akt-3 proteins.Alignment of all the previously described Akt sequences demonstratesthat Gly¹⁰ and Ala³⁹⁶ in the human protein correspond to Gly and Alaresidues respectively in the Akt-1 and Akt-2 sequences identified fromother species. Further evidence that we have identified the Akt-3isoform comes from the presence of isotype-specific sequencesrepresented by human Akt-3 residues 47–49 (LPY), 118–122 of SEQ ID NO: 3(NCSPT) and 139–141 (HHK). For each isotype, these sequences areconserved between species, but differ between the isotypes.

The human Akt-3 cDNA sequence was predicted to encode a NH₂-terminalpleckstrin homology (PH) domain (Musacchio et al., 1993) and aCOOH-terminal kinase domain. A striking difference between the human andrat Akt-3 protein sequence (Konishi, et al., 1995) is the presence of aCOOH-terminal Atail≅ comprising 74 residues after the kinase domain. Thelast 28 amino acid residues in human Akt-3 are absent from the rat Akt-3sequence. We were unable to identify human cDNA sequences that encoded asimilar truncation, despite conducting RACE experiments using cDNA fromseveral different human tissues. The region in human Akt-3 that isabsent from rat Akt-3 encompasses a stretch of 10 residues (residues467–476 in human Akt-3) which are identical to the corresponding regionof human Akt-1 and Akt-2. This suggests that the tail observed in humanAkt-3 is authentic. The significance of the difference observed in therat Akt-3 tail region remains to be investigated. However, the humanAkt-3 COOH-terminal sequence includes Ser⁴⁷², which corresponds toSer⁴⁷³ in Akt-1. Phosphorylation of Ser⁴⁷³ has been shown to lead to a5-fold increase in the activity of Akt-1, whereas a 20–25 fold increaseof Akt-1 activity is observed if both Ser⁴⁷³ and Thr³⁰⁸ arephosphorylated (Alessi et al., 1996). Thus, our observation that Ser⁴⁷²is present in human Akt-3 is significant, because it suggests that humanAkt-3 is potentially regulated in a manner similar to Akt-1 and Akt-2.Whether rat Akt-3 is regulated in a different fashion remains to beresolved.

The kinase and PH domains in Akt-3 show homology to the consensus PH andkinase domain sequences (Musacchio et al., 1993; Hanks & Hunter 1995).The PH domain of human Akt-3 is 77% and 86% identical to the PH domainsin Akt-1 and Akt-2, respectively, while the kinase domain of Akt-3 is88% and 87% identical to the kinase domain of Akt-1 and Akt-2,respectively. The high conservation of the PH domain may indicate anAkt-specific function, because PH domains are often highly divergent(Musacchio et al, 1993). Apart from binding phosphoinositides, the PHdomain of Akt has been shown to mediate interactions between Akt and PKC(Konishi, et al., 1995) as well as directing the formation of multimericAkt complexes (Datta et al, 1995). In contrast, the region between thePH domain and the kinase domain is poorly conserved between the humanAkt-1, Akt-2 and Akt-3 sequences, and this region is also important formediating the formation of multimeric Akt complexes (Datta et al, 1995).This raises an interesting issue—whether the sequence NH₂-terminal tothe kinase domain of Akt-3 mediates the interaction with bindingpartners that are unique to Akt-3 or that bind to multiple Akt isoforms.

To verify that the predicted kinase domain was catalytically active, weexpressed Akt-3 as a GST fusion protein in E. coli. The purified proteinwas able to phosphorylate an exogenous substrate, whereas no catalyticactivity was observed using GST in place of GST-Akt-3. To confirm thatAkt-3 is indeed regulated in a manner akin to Akt-1 and Akt-2, wemutated Thr³⁰⁵ and Ser⁴⁷³, either separately or jointly, to Asp. Thisstrategy has previously been shown to faithfully mimic the effect ofphosphorylation of these residues in Akt-1 (Alessi et al., 1996).Mutation of either of these residues resulted in increased activity,although the increase was less than that observed with Akt-1 (Alessi etal., 1996). Additionally, we did not observe a synergistic activation ofAkt-3 by mutation of both Thr³⁰⁵ and Ser⁴⁷³. In contrast, when both thecorresponding residues were simultaneously mutated to Asp in Akt-1,synergistic activation was observed (Alessi et al, 1996). The apparentquantitative differences between Akt-1 and Akt-3 may reflect truedifferences in the regulation of these two isoforms, or it may be due toother factors such as the different expression system used. In thepresent study Akt-3 was expressed as a GST fusion protein in E. coli,whereas Akt-1 activity was studied using an HA-tagged protein expressedin COS cells. Nevertheless, our results demonstrate that Akt-3 isqualitatively regulated in a fashion similar to Akt-1. Previous work hasalso shown that activation of Akt is dependent upon PI 3-kinase togenerate 3-phosphoinositides that bind the PH domain of Akt, promotetranslocation of Akt to the plasma membrane and facilitate thephosphorylation of Akt by upstream kinases (reviewed in Alessi & Cohen,1998; Coffer et al., 1998). Our observation that the T305D/S472D mutantof Akt-3 is more active than the wild type enzyme (FIG. 3), whenmeasured in the absence of 3-phosphoinositides, suggests that afterphosphorylation Akt-3 becomes (at least partially) independent ofphosphoinositide binding.

The structure of the catalytic domain of Akt is closely related toprotein kinase A and protein kinase C. Indeed, a BLAST search of theSwissProt data base revealed that the most closely related kinases(other than the different Akt isoforms) include several protein kinase Cisozymes. This prompted us to investigate whether existing inhibitors ofPKA or PKC, as well as other serine/threonine kinase inhibitors, couldbe used as inhibitors of Akt-3. Of the compounds tested, onlystaurosporine and the structurally related compound Ro 31-8220 bothpotently inhibited Akt-3. Staurosporine is a non-selective kinaseinhibitor, whereas Ro 31-8220 is a more selective PKC inhibitor (Davis,et al., 1992). Although Ro 31-8220 is an approximately 100-fold morepotent (IC₅₀.10 nM; Davis, et al., 1992) inhibitor of PKC than of Akt-3,this observation cautions that experiments using high concentrations ofRo 31-8820 may affect Akt-3. In contrast to staurosporine and Ro31-8220, two other PKC inhibitors and three other PKA inhibitors did notinhibit Akt-3. This suggests that although Akt-3 is closely related insequence to PKC, it may be possible to find selective inhibitors of Akt.

The observation that Akt-3 is activated by IGF-1 suggests that Akt-3 mayplay a role in regulating cell survival. Akt-3 potentially may suppressapoptosis in tumor cells. One concern in using Akt as a target for drugdevelopment in cancer is that Akt plays a role in insulin signalling(reviewed in Sheperd et al, 1998). Thus, inhibitors of Akt may inducesymptoms observed in patients with diabetes. One solution that has beenproposed is to develop selective inhibitors of Akt-2 (Walker et al,1998). This is based in part on the observation that Akt-1 is stronglyactivated by insulin in rat hepatocytes and skeletal muscle, whereasAkt-2 is only weakly activated by insulin in these tissues. However, ratAkt-3 appears to be even more weakly activated by insulin in thesetissues (Walker et al, 1998), and in this study we have shown that Akt-3mRNA is expressed only at low levels in human liver and skeletal muscle,which are insulin responsive tissues. This suggests that selectiveinhibitors of Akt-3 could have even less potential to cause symptomssimilar to those seen in patients with diabetes than do inhibitors ofAkt-2. The localisation of human Akt-3 to human chromosome 1q43–44 isalso interesting, as patients with haematological cancers have beenreported with chromosomal abnormalities in this region (Mitelman et al,1997). Although the significance of the latter observation is debatable,as chromosomal abnormalities at numerous loci have been observed inpatients with haematological cancers, the results presented hereindicate that Akt-3 may prove to be an important target for thedevelopment of novel therapeutics for the treatment of cancer.

Sequence Listing

-   1. Sequence ID No. 1 corresponds to the nucleotide sequence of Akt-3    illustrated in FIG. 1.-   2. Sequence ID No. 2 corresponds to from nucleotide position 11 to    1447 of the nucleic acid sequence of Akt-3 illustrated in FIG. 1.-   3. Sequence ID No. 3 corresponds to the amino acid sequence of Akt-3    illustrated in FIGS. 1 and 2.

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1. An isolated nucleic acid molecule encoding human Akt-3 protein,comprising the amino acid sequence illustrated in SEQ ID No.
 3. 2. Thenucleic acid molecule according to claim 1 which is a DNA molecule. 3.The nucleic acid molecule according to claim 1 comprising the nucleotidesequence illustrated in SEQ ID No.
 1. 4. The nucleic acid moleculeaccording to claim 1 comprising the nucleotide sequence in SEQ ID No. 2.5. An expression vector comprising the nucleic acid molecule accordingto claim
 2. 6. The expression vector according to claim 5 comprising aninducible promoter.
 7. The expression vector according to claim 5comprising a sequence encoding a reporter molecule.
 8. An isolated hostcell, transformed or transfected with the expression vector according toclaim
 5. 9. An isolated transgenic cell comprising the nucleic acidmolecule of claim 1, which expresses human Akt-3 protein as a transgene.